Evolution of the Biphenyl Dioxygenase BphA from Burkholderia xenovorans LB400 by Random Mutagenesis of Multiple Sites in Region III

Diane Barriault,Michel Sylvestre

doi:10.1074/jbc.m406805200

Abstract

It is now established that several amino acids of region III of the biphenyl dioxygenase (BPDO) alpha subunit are involved in substrate recognition and regiospecificity toward chlorobiphenyls. However, the sequence pattern of the amino acids of that segment of seven amino acids located in the C-terminal portion of the alpha subunit is rather limited in BPDOs of natural occurrence. In this work, we have randomly mutated simultaneously four residues (Thr(335)-Phe(336)-Ile(338)-Ile(341)) of region III of Burkholderia xenovorans LB400 BphA. The library was screened for variants able to oxygenate 2,2'-dichlorobiphenyl (2,2'-CB). Replacement of Phe(336) with Met or Ile with a concomitant change of Thr(335) to Ala created new variants that transformed 2,2'-CB into 3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl, which is a dead end metabolite that was not cleaved by BphC. Replacement of Thr(335)-Phe(336) with Ala(335)-Leu(336) did not cause this type of phenotypic change. Regiospecificity toward congeners other than 2,2'-CB that were oxygenated more efficiently by variant Ala(335)-Met(336) than by LB400 BPDO was similar for both enzymes. Thus structural changes that altered the regiospecificity toward 2,2'-CB did not affect the metabolite profile of other congeners, although it affected the rate of conversion of these congeners. It was especially noteworthy that both LB400 BPDO and the Ala(335)-Met(336) variant generated 2,3-dihydroxy-2',4,4'-trichlorobiphenyl as the sole metabolite from 2,4,2',4'-CB and 4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl as the major metabolite from 2,3,2',3'-CB. This shows that 2,4,2',4'-CB is oxygenated principally onto vicinal ortho-meta carbons 2 and 3 and that 2,3,2',3'-CB is oxygenated onto meta-para carbons 4 and 5 by both enzymes. The data suggest that interactions between the chlorine substitutes on the phenyl ring and specific amino acid residues of the protein influence the orientation of the phenyl ring inside the catalytic pocket.

Highlights

Biphenyl dioxygenase (BPDO)1 catalyzes the first reaction of the biphenyl catabolic pathway
It is established that several amino acids of region III of the biphenyl dioxygenase (BPDO) ␣ subunit are involved in substrate recognition and regiospecificity toward chlorobiphenyls
This shows that region III amino acids are not the only ones influencing the catalytic activity toward chlorobiphenyl, but it shows that the sequence pattern of region III of LB400 BphA is not optimal for catalytic activity toward chlorobiphenyls

Summary

Introduction

Biphenyl dioxygenase (BPDO) catalyzes the first reaction of the biphenyl catabolic pathway. This paper is available on line at http://www.jbc.org broader range of PCB congeners [6, 8, 9, 12] This shows that region III amino acids are not the only ones influencing the catalytic activity toward chlorobiphenyl, but it shows that the sequence pattern of region III of LB400 BphA is not optimal for catalytic activity toward chlorobiphenyls. The amino acid residues of region III BphA influence greatly the range of PCB congeners oxygenated by the enzyme, based on the alignment of sequences of BphAs that are found in data bases, the diversity of sequence patterns of this stretch of amino acids is rather limited [6]. The data suggest that despite the fact that residues 335 and 336 exert a strong influence on the regiospecificity toward 2,2Ј-CB and on the turnover rate toward several congeners, the pattern of chlorine substitution on the PCB substrate imposes its orientation toward the catalytic active center

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